For nonsynonymous coding variants, functional impact prediction algorithms frequently make use of conservation of amino acid substitutions observed among homologous proteins at a given site under the assumption substitutions occurring at well-conserved sites will deleteriously impact protein function and substitutions occurring at less conserved sites will be tolerated.

In this study, Marini et al. examine this assumption by evaluating a panel of nonsynonymous mutants in MTHFR using a yeast cell-based functional assay. As expected, substitutions in human MTHFR at sites that are well-conserved across distant orthologs result in an impaired enzyme. However, many substitutions that were tolerated by at least one ortholog of MTHFR proved deleterious to the human enzyme. The results suggest that selective constraints vary between phylogenetic lineages such that inclusion of distant orthologs to infer selective pressures on the human enzyme may be misleading.

Marini et al. suggest that as orthologs diverge from their most recent common ancestor, their different evolutionary trajectories lead to the divergence in the selective constraints on homologous sites. Therefore, in using amino acid conservation to predict for the function impact of genetic variation, homogenous proteins among only direct lineal ancestors of the particular protein of interest should be used.